Transcript
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form FOR RESEARCH USE ONLY
Date: __________________________ Illumina Kit Lot #: __________________________
Description: ______________________________________ _________________________________________________
NOTE Unless familiar with the LS protocol in the latest version of the TruSeq RNA Sample Preparation v2 Guide (part # 15026495), new or less experienced users are advised to follow the protocol in the guide before using this Experienced User Card and Lab Tracking Form.
ILLUMINA PROPRIETARY Catalog # RS-122-9002DOC Part # 15026498 Rev. F March 2014
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TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
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Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
Item
Lot Number
A-Tailing Control (CTA)
Lot #: _____________________
A-Tailing Mix (ATL)
Lot #: _____________________
Bead Binding Buffer (BBB)
Lot #: _____________________
Bead Washing Buffer (BWB)
Lot #: _____________________
Elute, Prime, Fragment Mix (EPF)
Lot #: _____________________
Elution Buffer (ELB)
Lot #: _____________________
End Repair Control (CTE)
Lot #: _____________________
End Repair Mix (ERP)
Lot #: _____________________
First Strand Master Mix (FSM)
Lot #: _____________________
Ligation Control (CTL)
Lot #: _____________________
Ligation Mix (LIG)
Lot #: _____________________
PCR Master Mix (PMM)
Lot #: _____________________
PCR Primer Cocktail (PPC)
Lot #: _____________________
Resuspension Buffer (RSB)
Lot #: _____________________
RNA Purification Beads (RPB)
Lot #: _____________________
Second Strand Master Mix (SSM)
Lot #: _____________________
Stop Ligation Buffer (STL)
Lot #: _____________________
80% Ethanol
Date Prepared: ____________
Part # 15026498 Rev. F
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Consumables
Consumables
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Consumables
Date/Time: _______________________________
Adapter Indexes
Operator: _______________________________
Lot Number
RNA Adapter Index 1 (AR001)
Lot #: _____________________
RNA Adapter Index 2 (AR002)
Lot #: _____________________
RNA Adapter Index 3 (AR003)
Lot #: _____________________
RNA Adapter Index 4 (AR004)
Lot #: _____________________
RNA Adapter Index 5 (AR005)
Lot #: _____________________
RNA Adapter Index 6 (AR006)
Lot #: _____________________
RNA Adapter Index 7 (AR007)
Lot #: _____________________
RNA Adapter Index 8 (AR008)
Lot #: _____________________
RNA Adapter Index 9 (AR009)
Lot #: _____________________
RNA Adapter Index 10 (AR010)
Lot #: _____________________
RNA Adapter Index 11 (AR011)
Lot #: _____________________
RNA Adapter Index 12 (AR012)
Lot #: _____________________
RNA Adapter Index 13 (AR013)
Lot #: _____________________
RNA Adapter Index 14 (AR014)
Lot #: _____________________
RNA Adapter Index 15 (AR015)
Lot #: _____________________
RNA Adapter Index 16 (AR016)
Lot #: _____________________
RNA Adapter Index 18 (AR018)
Lot #: _____________________
RNA Adapter Index 19 (AR019)
Lot #: _____________________
RNA Adapter Index 20 (AR020)
Lot #: _____________________
RNA Adapter Index 21 (AR021)
Lot #: _____________________
RNA Adapter Index 22 (AR022)
Lot #: _____________________
RNA Adapter Index 23 (AR023)
Lot #: _____________________
RNA Adapter Index 24 (AR024)
Lot #: _____________________
RNA Adapter Index 25 (AR025)
Lot #: _____________________
RNA Adapter Index 27 (AR027)
Lot #: _____________________
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Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
If you are pooling, use IEM or BaseSpace to record information about your samples before beginning library preparation. Illumina recommends arranging samples that will be combined into a common pool in the same row. Include a common index in each column. Sample Sheet Name: ________________________________________________
Comments __________________________________________________________________________________ __________________________________________________________________________________
Part # 15026498 Rev. F
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Prepare Adapter Setup
Prepare Adapter Setup
TruSeq RNA Sample Prep v2 LS Protocol
Prepare Adapter Setup
Experienced User Card and Lab Tracking Form
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Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
This process purifies the polyA containing mRNA molecules using oligo-dT attached magnetic beads using two rounds of purification. During the second elution of the polyA RNA, the RNA is also fragmented and primed for cDNA synthesis.
Consumables Item
Quantity
Storage
Supplied By
Bead Binding Buffer (BBB)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Bead Washing Buffer (BWB)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Elute, Prime, Fragment Mix (EPF)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Elution Buffer (ELB)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
-25°C to -15°C
Illumina
RNA Purification Beads (RPB)
1 tube per 48 reactions
2°C to 8°C
Illumina
RBP (RNA Bead Plate) barcode label
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plate
1
15°C to 30°C
User
Microseal ‘B’ adhesive seals
3
15°C to 30°C
User
RNase/DNase-free reagent reservoirs (if using multichannel pipettes)
6
15°C to 30°C
User
RNase/DNase-free eight-tube strips and caps (if using multichannel pipettes)
6
15°C to 30°C
User
Make RBP [_] 1
Dilute the total RNA with nuclease-free ultra pure water to a final volume of 50 µl in the new 96-well 0.3 ml PCR plate labeled with the RBP barcode.
[_] 2
Vortex the room temperature RNA Purification Beads tube vigorously to resuspend the oligo-dT beads.
[_] 3
Add 50 µl RNA Purification Beads to each well of the RBP plate to bind the polyA RNA to the oligo-dT beads. Gently pipette the entire volume up and down 6 times to mix thoroughly.
[_] 4
Seal the RBP plate with a Microseal ‘B’ adhesive seal.
Part # 15026498 Rev. F
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Purify and Fragment mRNA
Purify and Fragment mRNA
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Purify and Fragment mRNA
Date/Time: _______________________________
Operator: _______________________________
Incubate 1 RBP [_] 1
Place the sealed RBP plate on the pre-programmed thermal cycler. Close the lid and select mRNA Denaturation (65°C for 5 minutes, 4°C hold) to denature the RNA and facilitate binding of the polyA RNA to the beads.
[_] 2
Remove the RBP plate from the thermal cycler when it reaches 4°C.
[_] 3
Place the RBP plate on the bench and incubate at room temperature for 5 minutes to allow the RNA to bind to the beads. Start time: _____________________
Stop time: _____________________
Wash RBP [_] 1
Remove the adhesive seal from the RBP plate.
[_] 2
Place the RBP plate on the magnetic stand at room temperature for 5 minutes to separate the polyA RNA bound beads from the solution. Start time: _____________________
Stop time: _____________________
[_] 3
Remove and discard all of the supernatant from each well of the RBP plate.
[_] 4
Remove the RBP plate from the magnetic stand.
[_] 5
Wash the beads by adding 200 µl Bead Washing Buffer in each well of the RBP plate to remove unbound RNA. Gently pipette the entire volume up and down 6 times to mix thoroughly.
[_] 6
Place the RBP plate on the magnetic stand at room temperature for 5 minutes. Start time: _____________________
Stop time: _____________________
[_] 7
Centrifuge the thawed Elution Buffer at 600 × g for 5 seconds.
[_] 8
Remove and discard all of the supernatant from each well of the RBP plate.
[_] 9
Remove the RBP plate from the magnetic stand.
[_] 10 Add 50 µl Elution Buffer in each well of the RBP plate. Gently pipette the entire volume up and down 6 times to mix thoroughly. [_] 11 Seal the RBP plate with a Microseal ‘B’ adhesive seal. [_] 12 Store the Elution Buffer tube at 4°C.
Incubate 2 RBP [_] 1
Place the sealed RBP plate on the pre-programmed thermal cycler. Close the lid and select mRNA Elution 1 (80°C for 2 minutes, 25°C hold) to elute the mRNA from the beads.
[_] 2
Remove the RBP plate from the thermal cycler when it reaches 25°C.
[_] 3
Place the RBP plate on the bench at room temperature.
[_] 4
Remove the adhesive seal from the RBP plate.
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Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
[_] 1
Centrifuge the thawed Bead Binding Buffer at 600 × g for 5 seconds.
[_] 2
Add 50 µl Bead Binding Buffer to each well of the RBP plate. Gently pipette the entire volume up and down 6 times to mix thoroughly.
[_] 3
Incubate the RBP plate at room temperature for 5 minutes and store the Bead Binding Buffer tube at 2°C to 8°C. Start time: _____________________
[_] 4
Stop time: _____________________
Place the RBP plate on the magnetic stand at room temperature for 5 minutes. Start time: _____________________
Stop time: _____________________
[_] 5
Remove and discard all of the supernatant from each well of the RBP plate.
[_] 6
Remove the RBP plate from the magnetic stand.
[_] 7
Wash the beads by adding 200 µl Bead Washing Buffer in each well of the RBP plate. Gently pipette the entire volume up and down 6 times to mix thoroughly.
[_] 8
Store the Bead Washing Buffer tube at 2°C to 8°C.
[_] 9
Place the RBP plate on the magnetic stand at room temperature for 5 minutes. Start time: _____________________
Stop time: _____________________
[_] 10 Remove and discard all of the supernatant from each well of the RBP plate. [_] 11 Remove the RBP plate from the magnetic stand. [_] 12 Add 19.5 µl Elute, Prime, Fragment Mix to each well of the RBP plate. Gently pipette the entire volume up and down 6 times to mix thoroughly. [_] 13 Seal the RBP plate with a Microseal ‘B’ adhesive seal. [_] 14 Store the Elute, Prime, Fragment Mix tube at -25°C to -15°C.
Incubate RFP [_] 1
Place the sealed RBP plate on the pre-programmed thermal cycler. Close the lid and select Elution 2 - Frag - Prime (94°C for 8 minutes, 4°C hold) to elute, fragment, and prime the RNA.
[_] 2
Remove the RBP plate from the thermal cycler when it reaches 4°C and centrifuge briefly.
[_] 3
Proceed immediately to Synthesize First Strand cDNA on page 11.
Comments __________________________________________________________________________________ __________________________________________________________________________________
Part # 15026498 Rev. F
Page 9 of 34
Purify and Fragment mRNA
Make RFP
TruSeq RNA Sample Prep v2 LS Protocol
Purify and Fragment mRNA
Experienced User Card and Lab Tracking Form
Page 10 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
This process reverse transcribes the cleaved RNA fragments primed with random hexamers into first strand cDNA using reverse transcriptase and random primers.
Consumables Item
Quantity
Storage
Supplied By
First Strand Master Mix (FSM)
1 tube
-25°C to -15°C
Illumina
CDP (cDNA Plate) barcode label
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plate
1
15°C to 30°C
User
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
RNase/DNase-free reagent reservoirs (if using multichannel pipettes)
1
15°C to 30°C
User
RNase/DNase-free eight-tube strips and caps (if using multichannel pipettes)
1
15°C to 30°C
User
SuperScript II Reverse Transcriptase
1 tube
-25°C to -15°C
User
Make CDP [_] 1
Place the RBP plate on the magnetic stand at room temperature for 5 minutes. Do not remove the plate from the magnetic stand. Start time: _____________________
Stop time: _____________________
[_] 2
Remove the adhesive seal from the RBP plate.
[_] 3
Transfer 17 µl of the supernatant (fragmented and primed mRNA) from each well of the RBP plate to the corresponding well of the new 0.3 ml PCR plate labeled with the CDP barcode.
[_] 4
Centrifuge the thawed First Strand Master Mix tube at 600 × g for 5 seconds.
[_] 5
Add 50 µl SuperScript II to the First Strand Master Mix tube. If you are not using the entire contents of the First Strand Master Mix tube, add SuperScript II at a ratio of 1 µl SuperScript II for each 9 µl First Strand Master Mix. Mix gently, but thoroughly, and centrifuge briefly. Label the First Strand Master Mix tube to indicate that the SuperScript II has been added.
[_] 6
Add 8 µl First Strand Master Mix and SuperScript II mix to each well of the CDP plate. Gently pipette the entire volume up and down 6 times to mix thoroughly.
[_] 7
Seal the CDP plate with a Microseal ‘B’ adhesive seal and centrifuge briefly.
[_] 8
Return the First Strand Master Mix tube to -25°C to -15°C storage immediately after use.
Part # 15026498 Rev. F
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Synthesize First Strand cDNA
Synthesize First Strand cDNA
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Synthesize First Strand cDNA
Date/Time: _______________________________
Operator: _______________________________
Incubate 1 CDP [_] 1
Place the sealed CDP plate on the pre-programmed thermal cycler. Close the lid, and then select and run the 1st Strand program. [_] a Choose the pre-heat lid option and set to 100°C [_] b 25°C for 10 minutes [_] c 42°C for 50 minutes [_] d 70°C for 15 minutes [_] e Hold at 4°C
[_] 2
When the thermal cycler reaches 4°C, remove the CDP plate from the thermal cycler and proceed immediately to Synthesize Second Strand cDNA on page 13.
Comments __________________________________________________________________________________ __________________________________________________________________________________
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Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
This process removes the RNA template and synthesizes a replacement strand to generate ds cDNA. AMPure XP beads are used to separate the ds cDNA from the second strand reaction mix.
Consumables Item
Quantity
Storage
Supplied By
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Second Strand Master Mix (SSM)
1 tube per 48 reactions
-25°C to -15°C
Illumina
IMP (Insert Modification Plate) barcode label
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plate
1
15°C to 30°C
User
AMPure XP beads
90 µl per sample
2°C to 8°C
User
Freshly prepared 80% ethanol (EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
RNase/DNase-free reagent reservoir (if using multichannel pipettes)
4
15°C to 30°C
User
RNase/DNase-free eight-tube strips and caps (if using multichannel pipettes)
4
15°C to 30°C
User
Add SSM [_] 1
Centrifuge the thawed Second Strand Master Mix at 600 × g for 5 seconds.
[_] 2
Remove the adhesive seal from the CDP plate.
[_] 3
Add 25 µl thawed Second Strand Master Mix to each well of the CDP plate. Gently pipette the entire volume up and down 6 times to mix thoroughly.
[_] 4
Seal the CDP plate with a Microseal ‘B’ adhesive seal.
Incubate 2 CDP [_] 1
Place the sealed CDP plate on the pre-heated thermal cycler. Close the lid and incubate at 16°C for 1 hour. Start time: _____________________
Stop time: _____________________
[_] 2
Remove the CDP plate from the thermal cycler and place it on the bench.
[_] 3
Remove the adhesive seal from the CDP plate.
[_] 4
Let the CDP plate stand to bring it to room temperature.
Part # 15026498 Rev. F
Page 13 of 34
Synthesize Second Strand cDNA
Synthesize Second Strand cDNA
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Synthesize Second Strand cDNA
Date/Time: _______________________________
Operator: _______________________________
Purify CDP [_] 1
Vortex the AMPure XP beads until they are well dispersed.
[_] 2
Add 90 µl well-mixed AMPure XP beads to each well of the CDP plate containing 50 µl ds cDNA. Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 3
Incubate the CDP plate at room temperature for 15 minutes. Start time: _____________________
[_] 4
Stop time: _____________________
Place the CDP plate on the magnetic stand at room temperature, for 5 minutes to make sure that all of the beads are bound to the side of the wells. Start time: _____________________
Stop time: _____________________
[_] 5
Remove and discard 135 µl of the supernatant from each well of the CDP plate.
[_] 6
With the CDP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each well without disturbing the beads.
[_] 7
Incubate the CDP plate at room temperature for 30 seconds, and then remove and discard all of the supernatant from each well.
[_] 8
Repeat steps 6 and 7 one time for a total of two 80% EtOH washes.
[_] 9
Let the CDP plate stand at room temperature for 15 minutes to dry, and then remove the plate from the magnetic stand. Start time: _____________________
Stop time: _____________________
[_] 10 Centrifuge the thawed, room temperature Resuspension Buffer at 600 × g for 5 seconds. [_] 11 Add 52.5 µl Resuspension Buffer to each well of the CDP plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. [_] 12 Incubate the CDP plate at room temperature for 2 minutes. Start time: _____________________
Stop time: _____________________
[_] 13 Place the CDP plate on the magnetic stand at room temperature for 5 minutes. Start time: _____________________
Stop time: _____________________
[_] 14 Transfer 50 µl supernatant (ds cDNA) from the CDP plate to the new 96-well 0.3 ml PCR plate labeled with the IMP barcode. SAFE STOPPING POINT If you do not plan to proceed immediately to Perform End Repair on page 15, you can safely stop the protocol here. If you are stopping, seal the IMP plate with a Microseal ‘B’ adhesive seal and store at -25°C to -15°C for up to seven days.
Comments __________________________________________________________________________________ __________________________________________________________________________________
Page 14 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
This process converts the overhangs resulting from fragmentation into blunt ends using an End Repair Mix. The 3' to 5' exonuclease activity of this mix removes the 3' overhangs and the polymerase activity fills in the 5' overhangs.
Consumables Item
Quantity
Storage
Supplied By
End Repair Mix (ERP)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
[Optional] End Repair Control (CTE)
1 tube per 48 reactions
-25°C to -15°C
Illumina
ALP (Adapter Ligation Plate) barcode label
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plate
1
15°C to 30°C
User
AMPure XP beads
160 µl per sample
2°C to 8°C
User
Freshly prepared 80% ethanol (EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
RNase/DNase-free reagent reservoirs (if using multichannel pipettes)
5
15°C to 30°C
User
RNase/DNase-free eight-tube strips and caps (if using multichannel pipettes)
5
15°C to 30°C
User
Make IMP [_] 1
Do one of the following: • If using the in-line control reagent: — Centrifuge the thawed End Repair Control tube at 600 × g for 5 seconds. — Dilute the End Repair Control to 1/100 in Resuspension Buffer (1 µl End Repair Control + 99 µl Resuspension Buffer) before use. — Add 10 µl diluted End Repair Control to each well of the IMP plate that contains 50 µl ds cDNA. • If not using the in-line control reagent, add 10 µl Resuspension Buffer to each well of the IMP plate that contains 50 µl ds cDNA.
[_] 2
Add 40 µl End Repair Mix to each well of the IMP plate containing the ds cDNA. Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 3
Seal the IMP plate with a Microseal ‘B’ adhesive seal.
Part # 15026498 Rev. F
Page 15 of 34
Perform End Repair
Perform End Repair
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Perform End Repair
Date/Time: _______________________________
Operator: _______________________________
Incubate IMP [_] 1
Place the sealed IMP plate on the pre-heated thermal cycler. Close the lid and incubate at 30°C for 30 minutes. Start time: _____________________
[_] 2
Stop time: _____________________
Remove the IMP plate from the thermal cycler.
Clean Up IMP [_] 1
Remove the adhesive seal from the IMP plate.
[_] 2
Vortex the AMPure XP beads until they are well dispersed.
[_] 3
Add 160 µl well-mixed AMPure XP beads to each well of the IMP plate containing 100 µl End Repair Mix. Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 4
Incubate the IMP plate at room temperature for 15 minutes. Start time: _____________________
[_] 5
Stop time: _____________________
Place the IMP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. Start time: _____________________
Stop time: _____________________
[_] 6
Using a 200 µl single channel or multichannel pipette set to 127.5 µl, remove and discard 127.5 µl of supernatant from each well of the IMP plate.
[_] 7
Repeat step 6 one time.
[_] 8
With the IMP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each well without disturbing the beads.
[_] 9
Incubate the IMP plate at room temperature for 30 seconds, and then remove and discard all of the supernatant from each well.
[_] 10 Repeat steps 8 and 9 one time for a total of two 80% EtOH washes. [_] 11 Let the IMP plate stand at room temperature for 15 minutes to dry, and then remove the plate from the magnetic stand. Start time: _____________________
Stop time: _____________________
[_] 12 Add 17.5 µl Resuspension Buffer to each well of the IMP plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Start time: _____________________
Stop time: _____________________
[_] 13 Incubate the IMP plate at room temperature for 2 minutes. Start time: _____________________
Stop time: _____________________
[_] 14 Place the IMP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. Start time: _____________________
Stop time: _____________________
[_] 15 Transfer 15 µl of supernatant from each well of the IMP plate to the corresponding well of the new 0.3 ml PCR plate labeled with the ALP plate barcode.
Page 16 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
Comments __________________________________________________________________________________ __________________________________________________________________________________
Part # 15026498 Rev. F
Page 17 of 34
Perform End Repair
SAFE STOPPING POINT If you do not plan to proceed immediately to Adenylate 3' Ends on page 19, you can safely stop the protocol here. If you are stopping, seal the ALP plate with a Microseal ‘B’ adhesive seal and store at -25°C to -15°C for up to 7 days.
TruSeq RNA Sample Prep v2 LS Protocol
Perform End Repair
Experienced User Card and Lab Tracking Form
Page 18 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single ‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensures a low rate of chimera (concatenated template) formation.
Consumables Item
Quantity
Storage
Supplied By
A-Tailing Mix (ATL)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
[Optional] A-Tailing Control (CTA)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Ice bucket
As needed
-25°C to -15°C
User
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
RNase/DNase-free eight-tube strips and caps (if using multichannel pipettes)
3
15°C to 30°C
User
RNase/DNase-free reagent reservoirs (if using multichannel pipettes)
3
15°C to 30°C
User
Add ATL [_] 1
Do one of the following: • If using the in-line control reagent: — Centrifuge the thawed A-Tailing Control tube at 600 × g for 5 seconds. — Dilute the A-Tailing Control to 1/100 in Resuspension Buffer (For example, 1 µl A-Tailing Control + 99 µl Resuspension Buffer) before use. Discard the diluted A-Tailing Control after use. — Add 2.5 µl diluted A-Tailing Control to each well of the ALP plate. • If not using the in-line control reagent, add 2.5 µl Resuspension Buffer to each well of the ALP plate.
[_] 2
Add 12.5 µl thawed A-Tailing Mix to each well of the ALP plate. Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 3
Seal the ALP plate with a Microseal ‘B’ adhesive seal.
Part # 15026498 Rev. F
Page 19 of 34
Adenylate 3' Ends
Adenylate 3' Ends
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Adenylate 3' Ends
Date/Time: _______________________________
Operator: _______________________________
Incubate 1 ALP [_] 1
Place the sealed ALP plate on the pre-programmed thermal cycler. Close the lid, then select and run the ATAIL70 program. [_] a Choose the pre-heat lid option and set to 100°C [_] b 37°C for 30 minutes [_] c 70°C for 5 minutes [_] d Hold at 4°C
[_] 2
When the thermal cycler temperature is 4°C, remove the ALP plate from the thermal cycler, then proceed immediately to Ligate Adapters on page 21.
Comments __________________________________________________________________________________ __________________________________________________________________________________
Page 20 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
This process ligates multiple indexing adapters to the ends of the ds cDNA, preparing them for hybridization onto a flow cell.
Consumables Item
Quantity
Storage
Supplied By
Ligation Mix (LIG)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
RNA Adapter Indexes (AR001– AR016, AR018–AR023, AR025, AR027)
1 tube of each index being used, per column of 8 reactions
-25°C to -15°C
Illumina
Stop Ligation Buffer (STL)
1 tube per 48 reactions
-25°C to -15°C
Illumina
[Optional] Ligation Control (CTL)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Barcode labels for: • CAP (Clean Up ALP Plate) • PCR (Polymerase Chain Reaction)
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plates
2
15°C to 30°C
User
AMPure XP beads
92 µl per sample
2°C to 8°C
User
Freshly prepared 80% ethanol (EtOH)
800 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seals
3
15°C to 30°C
User
RNase/DNase-free reagent reservoirs (if using multichannel pipettes)
4–28
15°C to 30°C
User
RNase/DNase-free eight-tube strips and caps (if using multichannel pipettes)
4–28
15°C to 30°C
User
Add LIG [_] 1
Centrifuge the thawed RNA Adapter Index tubes, Ligation Control (if using Ligation Control), and Stop Ligation Buffer tubes at 600 × g for 5 seconds.
[_] 2
Immediately before use, remove the Ligation Mix tube from -25°C to -15°C storage.
[_] 3
Remove the adhesive seal from the ALP plate.
Part # 15026498 Rev. F
Page 21 of 34
Ligate Adapters
Ligate Adapters
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Ligate Adapters
Date/Time: _______________________________
Operator: _______________________________
[_] 4
Do one of the following: • If using the in-line control reagent: — Dilute the Ligation Control to 1/100 in Resuspension Buffer (1 µl Ligation Control + 99 µl Resuspension Buffer) before use. Discard the diluted Ligation Control after use. — Add 2.5 µl diluted Ligation Control to each well of the ALP plate. • If not using the in-line control reagent, add 2.5 µl Resuspension Buffer to each well of the ALP plate.
[_] 5
Add 2.5 µl Ligation Mix to each well of the ALP plate.
[_] 6
Return the Ligation Mix tube back to -25°C to -15°C storage immediately after use.
[_] 7
Add 2.5 µl thawed RNA Adapter Index to each well of the ALP plate. Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 8
Seal the ALP plate with a Microseal ‘B’ adhesive seal.
Incubate 2 ALP [_] 1
Place the sealed ALP plate on the pre-heated thermal cycler. Close the lid and incubate at 30°C for 10 minutes. Start time: _____________________
[_] 2
Stop time: _____________________
Remove the ALP plate from the thermal cycler.
Add STL [_] 1
Remove the adhesive seal from the ALP plate.
[_] 2
Add 5 µl Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation. Gently pipette the entire volume up and down 10 times to mix thoroughly.
Clean Up ALP [_] 1
Vortex the AMPure XP beads for at least 1 minute or until they are well dispersed.
[_] 2
Add 42 µl mixed AMPure XP beads to each well of the ALP plate. Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 3
Incubate the ALP plate at room temperature for 15 minutes. Start time: _____________________
[_] 4
Stop time: _____________________
Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. Start time: _____________________
Stop time: _____________________
[_] 5
Remove and discard 79.5 µl of supernatant from each well of the ALP plate.
[_] 6
With the ALP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each well without disturbing the beads.
[_] 7
Incubate the ALP plate at room temperature for 30 seconds, and then remove and discard all of the supernatant from each well.
[_] 8
Repeat steps 6 and 7 one time for a total of two 80% EtOH washes.
Page 22 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
With the ALP plate on the magnetic stand, let the samples air-dry at room temperature for 15 minutes. Start time: _____________________
Stop time: _____________________
[_] 10 Remove the ALP plate from the magnetic stand. [_] 11 Add 52.5 µl Resuspension Buffer to each well of the ALP plate. Gently pipette the entire volume up and down 10 times to mix thoroughly or until the beads are fully resuspended. [_] 12 Incubate the ALP plate at room temperature for 2 minutes. Start time: _____________________
Stop time: _____________________
[_] 13 Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. Start time: _____________________
Stop time: _____________________
[_] 14 Transfer 50 µl of supernatant from each well of the ALP plate to the corresponding well of the new 0.3 ml PCR plate labeled with the CAP barcode. [_] 15 Vortex the AMPure XP beads until they are well dispersed. [_] 16 Add 50 µl mixed AMPure XP beads to each well of the CAP plate for a second cleanup. Gently pipette the entire volume up and down 10 times to mix thoroughly. [_] 17 Incubate the CAP plate at room temperature for 15 minutes. Start time: _____________________
Stop time: _____________________
[_] 18 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. Start time: _____________________
Stop time: _____________________
[_] 19 Remove and discard 95 µl of supernatant from each well of the CAP plate. [_] 20 With the CAP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each well. [_] 21 Incubate the CAP plate at room temperature for 30 seconds, and then remove and discard all of the supernatant from each well. [_] 22 Repeat steps 20 and 21 one time for a total of two 80% EtOH washes. [_] 23 With the CAP plate on the magnetic stand, let the samples air-dry at room temperature for 15 minutes, and then remove the plate from the magnetic stand. Start time: _____________________
Stop time: _____________________
[_] 24 Add 22.5 µl Resuspension Buffer to each well of the CAP plate. Gently pipette the entire volume up and down 10 times to mix thoroughly or until the beads are fully resuspended. [_] 25 Incubate the CAP plate at room temperature for 2 minutes. Start time: _____________________
Stop time: _____________________
[_] 26 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. Start time: _____________________
Stop time: _____________________
[_] 27 Transfer 20 µl of supernatant from each well of the CAP plate to the corresponding well of the new 0.3 ml PCR plate labeled with the PCR barcode.
Part # 15026498 Rev. F
Page 23 of 34
Ligate Adapters
[_] 9
Operator: _______________________________
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Ligate Adapters
Date/Time: _______________________________
Operator: _______________________________
SAFE STOPPING POINT If you do not plan to proceed immediately to Enrich DNA Fragments on page 25, you can safely stop the protocol here. If you are stopping, seal the PCR plate with a Microseal ‘B’ adhesive seal and store at -25°C to -15°C for up to 7 days.
Comments __________________________________________________________________________________ __________________________________________________________________________________
Page 24 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
This process uses PCR to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. The PCR is performed with a PCR Primer Cocktail that anneals to the ends of the adapters. Minimize the number of PCR cycles to avoid skewing the representation of the library.
Consumables Item
Quantity
Storage
Supplied By
PCR Master Mix (PMM)
1 tube per 48 reactions
-25°C to -15°C
Illumina
PCR Primer Cocktail (PPC)
1 tube per 48 reactions
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
TSP1 (Target Sample Plate) barcode label
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plate
1
15°C to 30°C
User
AMPure XP beads
50 µl per sample
2°C to 8°C
User
Freshly prepared 80% ethanol (EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
Ice bucket
As needed
-25°C to -15°C
User
RNase/DNase-free eight-tube strips and caps (if using multichannel pipettes)
5
15°C to 30°C
User
RNase/DNase-free reagent reservoirs (if using multichannel pipettes)
5
15°C to 30°C
User
Make PCR [_] 1
Add 5 µl thawed PCR Primer Cocktail to each well of the PCR plate.
[_] 2
Add 25 µl thawed PCR Master Mix to each well of the PCR plate. Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 3
Seal the PCR plate with a Microseal ‘B’ adhesive seal.
Part # 15026498 Rev. F
Page 25 of 34
Enrich DNA Fragments
Enrich DNA Fragments
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Enrich DNA Fragments
Date/Time: _______________________________
Operator: _______________________________
Amp PCR [_] 1
Place the sealed PCR plate on the pre-programmed thermal cycler. Close the lid, then select and run PCR to amplify the plate. [_] a Choose the pre-heat lid option and set to 100°C [_] b 98°C for 30 seconds [_] c 15 cycles of: — 98°C for 10 seconds — 60°C for 30 seconds — 72°C for 30 seconds [_] d 72°C for 5 minutes [_] e Hold at 10°C
Clean Up PCR [_] 1
Remove the adhesive seal from the PCR plate.
[_] 2
Vortex the AMPure XP Beads until they are well dispersed.
[_] 3
Add 50 µl mixed AMPure XP Beads to each well of the PCR plate containing 50 µl of PCR amplified library. Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 4
Incubate the PCR plate at room temperature for 15 minutes. Start time: _____________________
[_] 5
Stop time: _____________________
Place the PCR plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. Start time: _____________________
Stop time: _____________________
[_] 6
Remove and discard 95 µl of supernatant from each well of the PCR plate.
[_] 7
With the PCR plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each well without disturbing the beads.
[_] 8
Incubate the PCR plate at room temperature for 30 seconds, and then remove and discard all of the supernatant from each well.
[_] 9
Repeat steps 7 and 8 one time for a total of two 80% EtOH washes.
[_] 10 With the PCR plate on the magnetic stand, let the samples air-dry at room temperature for 15 minutes and then remove the plate from the magnetic stand. Start time: _____________________
Stop time: _____________________
[_] 11 Resuspend the dried pellet in each well with 32.5 µl Resuspension Buffer. Gently pipette the entire volume up and down 10 times to mix thoroughly. [_] 12 Incubate the PCR plate at room temperature for 2 minutes. Start time: _____________________
Stop time: _____________________
[_] 13 Place the PCR plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. Start time: _____________________
Stop time: _____________________
Page 26 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
SAFE STOPPING POINT If you do not plan to proceed immediately to Validate Library on page 29, you can safely stop the protocol here. If you are stopping, seal the TSP1 plate with a Microseal ‘B’ adhesive seal and store at -25°C to -15°C for up to seven days.
Comments __________________________________________________________________________________ __________________________________________________________________________________
Part # 15026498 Rev. F
Page 27 of 34
Enrich DNA Fragments
[_] 14 Transfer 30 µl of clear supernatant from each well of the PCR plate to the corresponding well of the new 0.3 ml PCR plate labeled with the TSP1 barcode.
TruSeq RNA Sample Prep v2 LS Protocol
Enrich DNA Fragments
Experienced User Card and Lab Tracking Form
Page 28 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
Illumina recommends performing the following procedures for quality control analysis on your sample library and quantification of the DNA library templates.
Quantify Libraries Quantify your libraries using qPCR according to the Illumina Sequencing Library qPCR Quantification Guide (part # 11322363).
Quality Control [_] 1
Do one of the following: • Load 1 µl of resuspended construct on an Agilent Technologies 2100 Bioanalyzer using a DNA-specific chip such as the Agilent DNA 1000. • Dilute 1 µl of resuspended construct with 1 µl RSB and load on an Advanced Analytical Fragment Analyzer using Standard Sensitivity NGS Fragment Analysis Kit.
[_] 2
Check the size and purity of the sample. Check the size and purity of the sample. The final product should be a band at approximately 260 bp (for single-read libraries).
Comments __________________________________________________________________________________ __________________________________________________________________________________
Part # 15026498 Rev. F
Page 29 of 34
Validate Library
Validate Library
TruSeq RNA Sample Prep v2 LS Protocol
Validate Library
Experienced User Card and Lab Tracking Form
Page 30 of 34
Part # 15026498 Rev. F
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form Date/Time: _______________________________
Operator: _______________________________
This process describes how to prepare DNA templates for cluster generation. Indexed DNA libraries are normalized to 10 nM in the DCT plate and then pooled in equal volumes in the PDP plate. Non-indexed DNA libraries are normalized to 10 nM in the DCT plate.
Consumables Item
Quantity
Storage
Supplied By
Barcode labels for: • DCT (Diluted Cluster Template) • PDP (Pooled DCT Plate) (for pooling only)
1 label per plate
15°C to 30°C
Illumina
96-well MIDI plate
1
15°C to 30°C
User
96-well 0.3 ml PCR plate (for pooling only)
1
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
Tris-HCl 10 mM, pH8.5 with 0.1% Tween 20
Enough to normalize the concentration of each sample library to 10 nM
15°C to 30°C
User
Make DCT [_] 1
Transfer 10 µl of sample library from each well of the TSP1 plate to the corresponding well of the new MIDI plate labeled with the DCT barcode.
[_] 2
Normalize the concentration of sample library in each well of the DCT plate to 10 nM using Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20.
[_] 3
Gently pipette the entire normalized sample library volume up and down 10 times to mix thoroughly.
[_] 4
Depending on the type of library you want to generate, do one of the following: • For non-pooled libraries, the protocol stops here. Do one of the following: — Proceed to cluster generation. — Seal the DCT plate with a Microseal ‘B’ adhesive seal and store at -25°C to -15°C. • For pooled libraries, proceed to Make PDP (for pooling only).
Make PDP (for pooling only) [_] 1
Determine the number of samples to be combined together for each pool.
[_] 2
Transfer 10 µl of each normalized sample library to be pooled from the DCT plate to one well of the new 0.3 ml PCR plate labeled with the PDP barcode. The total volume in each well of the PDP plate is 10X the number of combined sample libraries and 20–240 µl (2–24 libraries).
Part # 15026498 Rev. F
Page 31 of 34
Normalize and Pool Libraries
Normalize and Pool Libraries
TruSeq RNA Sample Prep v2 LS Protocol Experienced User Card and Lab Tracking Form
Normalize and Pool Libraries
Date/Time: _______________________________
Operator: _______________________________
[_] 3
Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 4
Do one of the following: • Proceed to cluster generation. • Seal the PDP plate with a Microseal ‘B’ adhesive seal and store at -25°C to -15°C.
Comments __________________________________________________________________________________ __________________________________________________________________________________
Page 32 of 34
Part # 15026498 Rev. F
Technical Assistance For technical assistance, contact Illumina Technical Support. Table 1 Illumina General Contact Information Illumina Website Email
www.illumina.com
[email protected]
Table 2 Illumina Customer Support Telephone Numbers Region Contact Number Region North America 1.800.809.4566 Italy Austria 0800.296575 Netherlands Belgium 0800.81102 Norway Denmark 80882346 Spain Finland 0800.918363 Sweden France 0800.911850 Switzerland Germany 0800.180.8994 United Kingdom Ireland 1.800.812949 Other countries
Contact Number 800.874909 0800.0223859 800.16836 900.812168 020790181 0800.563118 0800.917.0041 +44.1799.534000
Safety Data Sheets Safety data sheets (SDSs) are available on the Illumina website at www.illumina.com/msds.
Product Documentation Product documentation in PDF is available for download from the Illumina website. Go to www.illumina.com/support, select a product, then click Documentation & Literature.
Part # 15026498 Rev. F
Page 33 of 34
*15026498* Part # 15026498 Rev. F
Illumina San Diego, California 92122 U.S.A. +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America)
[email protected] www.illumina.com
